Process of selective bioconversion of isosorbitol dinitrate

ABSTRACT

The invention relates to a process of selective bioconversion of DINIS into MONIS-5, wherein the DINIS is added to a medium containing at least one microorganism possessing a glutathion transferase or an acellular extract of such a microorganism.

The present invention relates to the selective bioconversion of nitric ester functions into hydroxy functions of the dinitrate of isosorbitol (DINIS).

Distinction is made between two mononitrates of isosorbitol which are named MONIS. When the hydroxy function is in exo position, the compound is named MONIS-5, and when this function is in the endo position, MONIS-2. These two compounds may be represented as follows: ##STR1##

The MONIS are known as coronary vasodilators after the fashion of the dinitrate of isosorbitol (DINIS) and of nitroglycerin.

The MONIS are prepared at present by processes which have drawbacks. Synthesis by nitration of isosorbitol requires the use of a nitration mixture HNO₃ --acetic anhydride--acetic acid which can lead to the formation of acetyl nitrate considered as an explosive more sensitive than nitroglycerin.

Another process consists of preparing the acetates of isosorbitol and then nitrating them; here again, the risks of explosion are considerable, the process long and the yield low.

Novel processes have enabled the risks to be limited in the preparation of this type of compound but without however being fully satisfactory from the point of view of safety and/or yield, as will be seen from French patent 2 500 835.

This is why gentle techniques of preparing these compounds employing bioconversions have great interest.

The process according to the present invention is more particularly intended for the selective bioconversion of DINIS to MONIS-5, and is characterized in that the DINIS is added to a medium containing at least one microorganism possessing a glutathion transferase or an acellular extract of such a microorganism, preferably also the medium involved will be a culture medium for the microorganism.

Among the microorganisms usable within the scope of the present invention, must be mentioned more particularly the protozoa, fungi and bacteria. Among the protozoa, must be mentioned more particularly Tetrahymena, and among the fungi, must be mentioned more particularly Beauveria, Aspergillus, Streptomyces and Cunninghamella.

The fermentation of these microorganisms in a medium containing DINIS leads to the formation of MONIS, with a preponderance of MONIS-5 with respect to MONIS-2.

Although the bioconversion of DINIS may be effected in a medium only containing microorganism cells, it is preferable to effect this bioconversion in a culture medium. This culture medium will, preferably, be a minium medium containing, preferably, 5 to 20 g/l of glucose, yeast extracts, and the bioconversion will be carried out at a temperature of the order of 20° to 30° C. at a pH close to neutrality.

Experiments carried out have enabled it to be shown that the addition of amino acids to the culture medium increased distinctly the yield of MONIS-5.

Under the preceding conditions, a concentration of a DINIS comprised between 1 and 5 g/l is utilisable.

It is, in addition, preferable to use, when it is possible for the fungis, an already aged mycelium which gives higher bioconversion yields. Of course, when a growth medium is used, it is necessary to provide for aeration of the medium.

As has been previously indicated, it is also possible to provide for the use of the mycelium in a medium which is not a growth medium; it is even possible to use, in certain cases, extracts partly freed from a cellular structure, this facilitating the subsequent separating operations.

In certain cases, the microorganisms could be used in immobilized form, for example by absorption on a support or by encapsulation in a polymer, agar for example.

The conditions of practicing this bioconversion enables any risk associated with manipulation of this type of product to be avoided and enable yields of MONIS-5 to be obtained which are very considerable, as will emerge from the following examples.

The DINIS used within the scope of the present invention by way of starting material is an explosive substance, but it is possible to handle it with complete safety to the extent that this substance is moist or impregnated with solvent or in solution.

The following examples are intended to demonstrate other advantages and features of the present invention.

EXAMPLE 1 Bioconversion of DINIS by cultures of Cunninghemella echinulata (C. ech.) NRRL 3655 and Cunninghamella elegans (C. el.) ATCC 3245

The precultures were made in an inclined agar tube, at 24° on potato gelose (I.P.P.). The cultures (50 ml) were grown in "soya flour" liquid medium in Erlenmeyer flasks of 250 ml, shaken (180 rpm.) at 24° C. The seedings were preformed by the addition of 0.5 to 1 ml of a suspension of spores at the average density of 3.10⁵ spores/ml. After 44 hours of growth, and according to the test, the DINIS was added either to the culture, or to the mycelium suspension in phosphate buffer 0.05M pH 7, and incubated at 24° C. for selected periods of time.

Various conditions of biotransformation were studied:

a) Incubation in a culture medium, the DINIS is added at 1 g/l in solution in methanol (50 mg/ml).

b) Incubation in phosphate buffer 0.05M pH 7, the DINIS is added at 1 g/l in solution in methanol (50 mg/ml).

c) Incubation in phosphate buffer 0.05M pH 7, the DINIS is added in powder at 1 g/l.

The amounts of MONIS-5 and MONIS-2 formed in the course of time by bioconversion, were estimated by analysis by HPLC on a μBondapack C-18 column.

The results obtained are collected for C. ech. in Table 1 and for C. el. in Table 2.

                                      TABLE 1                                      __________________________________________________________________________     Biotransformation of DINIS by Cunninghamella echinulata                        NRRL 3655 (ATCC 36190)                                                         __________________________________________________________________________     (a) Incubation in culture medium; DINIS 1 g/l in methanol                      (50 mg/ml); age of the cultures 44 hours; amount of                            mycelium: 73 g/l;                                                              Hours     4  8  21 28 33 46 55 73 100                                          __________________________________________________________________________     DINIS (mg/l)                                                                             595                                                                               570                                                                               535                                                                               473                                                                               445                                                                               351                                                                               339                                                                               262                                                                               182                                          MONIS-2                                                                              (mg/l)                                                                             8  26 56 66 73 83 100                                                                               104                                                                               103                                          MONIS-5                                                                              (mg/l)                                                                             27 64 134                                                                               175                                                                               186                                                                               232                                                                               239                                                                               268                                                                               197                                          __________________________________________________________________________     (b) Incubation in phosphate buffer 0.05 M, pH 7; DINIS                         1 g/l in methanol (50 mg/l); age of cultures 44                                hours; amount of mycelium: 80 g/l;                                             Hours     4  8  21 33 46 73 120                                                __________________________________________________________________________     DINIS (mg/l)                                                                             507                                                                               655                                                                               442                                                                               443                                                                               394                                                                               328                                                                               272                                                MONIS-2                                                                              (mg/l)                                                                             10 24 27 62 76 83 103                                                MONIS-5                                                                              (mg/l)                                                                             26 51 108                                                                               148                                                                               198                                                                               209                                                                               227                                                __________________________________________________________________________     (c) Incubation in phosphate buffer 0.05 M, pH 7; DINIS                         1 g/l in powder; age of the cultures 44 hours;                                 amount of mycelium 101 g/l;                                                    Hours     8  21 33 46 73                                                       __________________________________________________________________________     DINIS (mg/l)                                                                             605                                                                               533                                                                               510                                                                               480                                                                               406                                                      MONIS-2                                                                              (mg/l)                                                                             15 42 48 56 82                                                       MONIS-5                                                                              (mg/l)                                                                             30 64 101                                                                               136                                                                               145                                                      __________________________________________________________________________      Initial concentration of DINIS: The concentrations evaluated in DINIS,         MONIS2 and MONIS5 are not corrected. (An evaporation of the order of 10 t      15% must be taken into account).                                         

                  TABLE 2                                                          ______________________________________                                         Biotransformation of DINIS by Cunninghamella                                   elegans (ATCC 9245)                                                            ______________________________________                                         (a) Incubation in culture medium; DINIS 1 g/l in meth-                         anol (50 mg/ml); age of the cultures: 44 hours;                                amount of mycelium: 73 g/l;                                                              Hours                                                                                4      21   33   46   55   73   120                            ______________________________________                                         DINIS   (mg/l)  678    545  441  242  265  123  14,5                           MONIS-2 (mg/l)  18     112  165  185  314  305  170                            MONIS-5 (mg/l)  26     99   121  160  214  230  188                            ______________________________________                                         (b) Incubation in a phosphate buffer 0.05 M, pH 7;                             DINIS 1 g/l in methanol (50 mg/ml); age of the cul-                            tures: 44 hours; amount of mycelium: 80 g/l;                                             Hours                                                                                4       8    21    33   46    73                               ______________________________________                                         DINIS   (mg/l)  631     625  517   463  344   325                              MONIS-2 (mg/l)  13      19   63    98   115   149                              MONIS-5 (mg/l)  23      32   71    102  115   126                              ______________________________________                                          Initial concentration of DINIS: The concentrations evaluated in DINIS,         MONIS2 and MONIS5 are not corrected. (An evaporation of the order of 10 t      15% must be taken into account.                                          

1. The two strains C. ech. and C. el. convert the DINIS into MONIS-5 and MONIS-2.

2. The best conversion ratio is observed when the strain C. ech. is incubated in a culture medium for 73 hours. An extended incubation time leads to the drop of the total yield (DINIS+MONIS-5+MONIS-2) apparently by subsequent conversion of mononitrated derivatives into isosorbide.

Under these conditions, the mixture DINIS, MONIS-5, MONIS-2 is obtained with a total yield of 58%. The mononitrated derivatives are formed with respective yields of 33% of MONIS-5 and 13% approximately of MONIS-2 (yield calculated on the amount of DINIS consumed), namely a ratio: ##EQU1##

In order to improve the conversion yields, tests were carried out on an amount of DINIS reduced to 500 mg/l; the results obtained are collected in Table 3 below:

                  TABLE 3                                                          ______________________________________                                         Biotransformation of DINIS by Cunninghamella                                   echinulata NRRL 3655 (ATCC 36190)                                              Hours       Test    48     69    93   116   140                                ______________________________________                                         DINIS   (mg/l)* 1       268  169   107  68    60                                               2       270  169   116  86    75                               MONIS-5 (mg/l)* 1       143  190   216  218   244                                              2       150  197   219  225   238                              MONIS-2 (mg/l)* 1       61   67    95   71    93                                               2       58   89    98   106   96                               ______________________________________                                         DINIS 500 mg/l in methanol (50 mg/ml); incubation in                           culture medium; age of the cultures: 44 hours; aver-                           age amount of mycelium: 78 g/l;                                                ______________________________________                                          *The concentrations evaluated in DINIS, MONIS5 and MONIS2 are not              corrected. (Evaporation of the order of 10 to 15% must be taken into           account).                                                                

Under these conditions, it is possible to obtain, for incubation periods varying from 63 to 93 hours, the mixture (DINIS+MONIS-5+MONIS-2) with an overall yield of 80-90%. The mononitrated derivatives MONIS-5 and MONIS-2 are formed with respective yields of 50 and 22% (calculated on the amount of DINIS consumed).

Contrary to strain C. ech. which leads to the preferential formation of MONIS-5, the strain C. el. performs the conversion of the DINIS preferentially into isomer 2.

These results indicate that in a same family of Phycomycetes, the different strains can contain, in variable proportions, enzymes which specifically must enable the conversion of the DINIS into each of the isomers MONIS-5 and MONIS-2.

EXAMPLE 2 Mutagenesis trials of Cunninghamella echinulata

Development of a mutagenesis with aziridine (ethyleneimine)

After various attempts, it has appeared that the treatment of C. ech. spores for 4 minutes with a lethal solution of 5% aziridine induced the formation of mutants detectible after 72 hours of incubation on gelose.

Mutagenesis conditions

The spores of C. ech. sampled from 2 inclined tubes of pre-cultures were washed with 8 ml of water (4 ml/tube) and resuspended in 4.75 ml of water to which 0.25 ml of aziridine (final solution 5%) were added.

After 4 minutes specimens (0.5 ml) were taken up, diluted (10², 10³, 10⁴ times) then seeded on Petri dishes of potato gelose in the proportion of 0.2 ml/dish.

The results obtained show that:

1. The concentration in spores of the solution before treatment was 2×10⁵ spores/ml and 3×10³ spores/ml (1.5%) after treatment.

2. Possibility of isolating muted strains after 72 hours of cultures.

The effectiveness of the mutants isolated to effect the biotransformation of the DINIS was evaluated in a liquid medium under conventional conditions.

Results

15 mutants were isolated and cultivated on inclined agar. The mutants were evaluated for their capacity to biotransform DINIS, under the conditions selected for C. ech. (44-hour mycelium). The biotransformation was evaluated after 48 hours of incubation.

It appears:

1. That it is possible to vary in the strains, by mutagenesis, the proportion of bioconversion of MONIS-2 and MONIS-5 such that the ratio MONIS-5/MONIS-2 varies from 2.07 to 2.91, hence the ratio of the enzymes responsible for the bioconversion.

2. That 2 mutants are more effective than the initial strain to obtain preferentially the MONIS-5 from DINIS, a mutant resulting preferentially in isomer 2.

3. That two mutants effect the bioconversion with a quantitative overall yield (DINIS+MONIS-5+MONIS-2)

EXAMPLE 3

Bioconversion of DINIS by various strains of Beauveria, Aspergillus and Streptomyces

The following experiments were carried out with various fungi. These were:

Beauveria bassiana ATCC 7159

Aspergillus ochraceus ATCC 18500

Aspergillus foetidus ATCC 10254

Aspergillus allicaeus ATCC 10060

Aspergillus niger ATCC 9142

Stroptomyces Tu 96

Streptomyces incarnatus.

The preliminary experiments which are reported were made under the conditions used for C. ech. (Example 1).

The precultures were carried out on inclined potato gelose. The cultures were grown in a "soya flour" liquid medium for 48 hours. The DINIS was added (500 mg/l) to the culture medium and incubated for 44 and 68 hours. The bioconversion ratios were obtained by determination by HPLC.

The results obtained (Table 4) indicate that:

1. All the strains studied performed the conversion of the DINIS into MONIS-5 and MONIS-2 in variable proportions and at variable speeds.

2. The Beauveria bassiana ATCC 7159 strain is noteworthy; it results after 48 hours of incubation in a complete and quantitative conversion of the DINIS into MONIS-5 and MONIS-2 with respective yields of 85 and 15%.

                  TABLE 4                                                          ______________________________________                                         Biotransformation of DINIS by different                                        strains of fungi                                                                      Duration of                                                                    incubation                                                                              Amount of substance                                            Strain   (hour)     MONIS-2   MONIS-5  DINIS                                   ______________________________________                                         Beauveria                                                                               44         46        252      110                                     bassiana 68         66        361       0                                      ATCC 7159                                                                      Aspergillus                                                                             44         18        31       430                                     foetidus 68         23        38       425                                     ATCC 10254                                                                     Aspergillus                                                                             44         60        71       286                                     alliaiens                                                                               68         61        97       212                                     ATCC 10060                                                                     Aspergillus                                                                             44         0         43       383                                     niger    68         10        58       377                                     ATCC 9142                                                                      Aspergillus                                                                             44         0         27       396                                     ochraceus                                                                               68         0         38       373                                     ATCC 18500                                                                     Streptomyces                                                                            44         0         21       497                                     Tu 96    68         28        47       413                                     Streptomyces                                                                            44         20        70       400                                     iscariatus                                                                              68         53        100      360                                     ______________________________________                                          The DINIS (500 mg/ml) was added to the culture after 48 hours of growth.       The evaluation of the yield takes into account the loss of the NO.sub.2        group during the conversion DINIS (MW 236) into MONIS (MW 191).          

EXAMPLE 4

1) Culture conditions of the strain Beauveria bassiana (ATCC 7159)

1.1) Pre-cultures

The pre-cultures were carried out at 24° C. over 6 days in an agar tube on 2 media at choice:

potato gelose (I.P.P.);

M₂ medium constituted of "Bacto Yeast extract" 4 g, "Bacto Malt extract" 10 g, "Bacto Dextrose" 4 g, agar 20 g per 1 liter of water, pH 7, according to E. B. Shirling and D. Gottlieb (Intern. J. Syst. Bact. 16, 313-340, 1966).

A more abundant sporulation is observed on M₂ medium.

1.2) Cultures

The cultures of B. bassiana (B. b.) were carried out in "soya flour" liquid medium according to R. E. Betts, D. E. Walters, J. P. Rossaza (J. Med. Chem. 17, 597-602, 1974), constituted by soya flour 5 g, glucose 20 g. "Yeast extract" 5 g, NaCl 5 g, K₂ HPO₄ 5 g, mineral salts Fe⁺² 0.1 mg/l, Zn⁺² 0.1 mg/l, Mo⁺² 0.01 mg/l, Cu⁺² 0.02 mg/l, Mn⁺² 0.01 mg/l, water at pH 7.

50 ml medium was seeded with 0.5 to 1 ml of a spore suspension 500 000 to 300 000 spores/ml) prepared from an agar tube taken up again with 6 ml of distilled water. The cultures carried out in Erlenmeyer flasks of 250 ml were shaken on a rotary shaker at 180 rpm at 24° C.

2) Analysis of the biotransformation products

The relative concentrations of DINIS, MONIS-5 and MONIS-2 were evaluated at HPLC.

Analysis was carried out on a Waters apparatus equipped with a U.V. detector (Pye Unicam) and a Waters integrater.

μBondapack C18 (10 μm-4×250 mm) column

solvent MeOH/H₂ O:25/75

flow rate 0.5 ml/min.

U.V. detector λ=220 nm, sensitivity 0.08/5.

The separation of the 3 compounds is satisfactory:

MONIS-5:Rt=10 min 20 sec

MONIS-2:Rt=9 min

DINIS:Rt=25 min 20 sec

The very great sensitivity of the technique enables the evaluation of amounts of substances of the order of 60 ng (3 mg/l) to 6 μg (300 mg/l) per 20 μg injected, hence concentrations compatible with the conditions used in the bioconversion trials. The linear response obtained for the three compounds between the surface of the peaks and the amounts of substances injected enables the evaluation of the concentrations of the different constituents present in the incubation medium.

3) Extraction of the constituents from the culture medium

0.5 ml of culture medium after filtration (or centrifugation) of the mycelium is placed on a glass column (diameter of 0.5 cm) filled with 1 g of Kieselgel 60 silica. 10 ml of ethyl ether were passed through. The ether was evaporated to dryness under vaccuum, and the sample was diluted with 2 ml of the mixture MeOH/H₂ O (25/75). This solution was injected directly into an HPLC apparatus. The extraction was quantitative.

4) Study of the bioconversion conditions of the DINIS by Beauveria bassiana

Studies were carried out systematically on cultures of 50 ml in volume, aged 48 hours, the DINIS was added in solution in methanol at 50 mg/ml.

4.1) Incubation in a culture medium:importance of the concentration of the DINIS (0.5 and 1 g/l)

The bioconversion was evaluated between 17 and 45 hours of incubation, the results obtained with a concentration of DINIS at 0.5 g/l (Table 5) indicate an almost complete bioconversion from 25 hours (residual amount of DINIS 3%), and total at 38 hours, which leads to the formation of the mixture MONIS-5 (85%) and MONIS-2 (15%), namely a ratio MONIS-5/MONIS-2=5.16/1, with a yield of 93%.

                  TABLE 5                                                          ______________________________________                                         Biotransformation of DINIS by Beauveria                                        bassiana ATCC 7159                                                             Time     Amount of substance                                                   (hour)   MONIS-5       MONIS-2   DINIS                                         ______________________________________                                         17       108           33        361                                           18       190           48        304                                           25       307           62        15                                            38       315           61        0                                             45       290           59        0                                             ______________________________________                                          Incubation of B. bassiana in culture medium. Concentration of the DINIS:       500 mg/ml.                                                               

When the concentration of DINIS is brought to 1 g/l for the same amount of Mycelium (Table 6), 68 hours of incubation are necessary to observe complete consumtion of the DINIS, formation of the MONIS-2, MONIS- is effected with an overall yield of 91%. However this slower bioconversion leads to the mixture MONIS-5/MONIS-2=3.9/1, hence less favorable to the production of the isomer 5.

                  TABLE 6                                                          ______________________________________                                         Biotransformation of DINIS by Beauveria                                        bassiana ATCC 7159                                                             Time     Amount of substance                                                   (hour)   MONIS-5       MONIS-2   DINIS                                         ______________________________________                                         17       91            41        851                                           45       449           138       438                                           68       588           150       0                                             ______________________________________                                          Incubation of B. bassiana in a culture medium. Concentration of the DINIS      1 g/l                                                                    

The importance of the ratio amount of DINIS/amount of mycelium for the selectivity of the biotransformation is illustrated by these experiments.

4.2) Incubation in water

A second series of experiments was performed in water. These conditions should permit a simplified extraction of the bioconversion products from the incubation medium devoid of the constituents of the culture medium.

A mycelium obtained from a 48 hour culture was suspended in water in the presence of a methanol solution of DINIS at the final concentration of 1 g/l.

Analysis of the bioconversion medium (Table 7) shows that under these conditions B. bassiana very slowly hydrolyzes DINIS. After 30 hours incubation, 40% of DINIS is still present in the medium.

                  TABLE 7                                                          ______________________________________                                         Biotransformation of DINIS by Beauveria                                        bassiana ATCC 7159                                                             Time     Amount of substance                                                   (hour)   MONIS-5       MONIS-2   DINIS                                         ______________________________________                                         17       48            21        987                                           45       171           44        767                                           68       330           91        600                                           90       432           100       10                                            ______________________________________                                          Incubation of B. bassiana in water. DINIS: 1 g/l                          

We claim:
 1. A process of selective bioconversion of dinitrate of isosorbitol (DINIS) into exo mononitrate of isosorbitol (MONIS-5) having the formula ##STR2## wherein said DINIS is provided in a culture medium containing at least one microorganism possessing a glutathion transferase or an acellular extract of such a microorganism.
 2. A process according to claim 1, wherein the DINIS is placed in a culture medium containing a microorganism possessing a glutathion transferase.
 3. A process according to claim 1, wherein the microorganism is selected from among protozoa, fungi and bacteria.
 4. A process according to claim 3, wherein the microorganism is selected from the types: Tetrahymena, Cunninghamella, Aspergillus, Beauveria and Streptomyces.
 5. A process according to claim 1, wherein the microorganism is selected from the genus Beauveria.
 6. A process according to claim 1, wherein the bioconversion is carried out at a temperature of the order of 20° to 30° C.
 7. A process according to claim 1, wherein the medium is a minimum culture containing from 5 to 20 g/l of glucose and a source of organic nitrogen containing amino acids.
 8. A process according to claim 1, wherein the DINIS is provided in a concentration of 1 to 5 g/l of medium.
 9. A process according to claim 1, wherein the pH of the medium is close to neutrality. 